Staphylococcal community of a small unit manufacturing traditional dry fermented sausages
Introduction
Traditional dry sausages are essentially made with pork lean and fat that are mixed with salt, spices and are processed without the addition of starter cultures. The fermentation of these products only relies on the indigenous microbial flora whose composition is variable and the growth promoted by the environmental conditions (Cocolin et al., 2001a, Rebecchi et al., 1998). This indigenous flora is composed by a wide variety of micro-organisms derived from raw materials contaminated during slaughter. This flora could colonize the batter, the products or the environment of the small processing units (Larrouture et al., 2000, Talon et al., 2003). The micro-organisms of technological interest isolated from the indigenous flora of naturally fermented sausages generally belong to the Lactic Acid Bacteria (LAB) group and to the Gram-positive Catalase-positive Cocci (GCC+) group mainly represented by the Staphylococcus and Kocuria genera. The LAB ensure the stability of the products mainly by producing lactic acid, which prevents the growth of pathogens. The GCC+ are involved in color development thanks to the nitrate reductase activity and enhance the sensory properties of fermented sausages mainly through the amino and fatty acid degradations (Talon et al., 1999, Talon et al., 2002).
Among the GCC+, many staphylococcal species have been isolated from dry fermented sausages. The Staphylococcus xylosus and S. saprophyticus species were found dominant in Greek fermented sausages (Samelis et al., 1998, Papamanoli et al., 2002, Drosinos et al., 2005). In Spanish sausages, S. xylosus was the dominant species (Garcia-Varona et al., 2000) whereas in low-acid chorizos the staphylococcal flora was more diversified, dominated by S. xylosus, S. carnosus and S. epidermidis (Aymerich et al., 2003). In Italian sausages, the indigenous flora was composed of S. xylosus, S. saprophyticus and S. equorum but many other species have been identified such as S. succinus, S. warneri, S. vitulinus, S. pasteuri, S. epidermidis, S. lentus and S. haemolyticus (Coppola et al., 2000, Cocolin et al., 2001a, Rossi et al., 2001). In traditional French dry fermented sausages, the main species identified were S. xylosus, S. carnosus, S. warneri and S. saprophyticus (Montel et al., 1992, Montel et al., 1996).
The identification of staphylococci to the species level by phenotypical methods has limitations and may have resulted in misidentifications (Rhoden and Miller, 1995, Blaiotta et al., 2003). In order to provide increasingly reliable identifications, several molecular methods have been developed, including PCR-based methods such as randomly amplified polymorphic DNA-PCR analysis (Rossi et al., 2001), species-specific PCR (Aymerich et al., 2003, Morot-Bizot et al., 2003), multiplex PCR (Corbière Morot-Bizot et al., 2004), partial sequencing of the highly conserved tuf, sodA or hsp60 genes for the identification of species of the genus Staphylococcus (Martineau et al., 2001, Poyart et al., 2001, Kwok and Chow, 2003), and oligonucleotide array targeting sodA gene (Giammarinaro et al., 2005). Many typing methods have also been used to characterize staphylococci, such as pulse-field gel electrophoresis (PFGE) (Snopkova et al., 1994), ribotyping (Villard et al., 2000), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments (Cocolin et al., 2001b) and amplification of the 16S-23S intergenic spacer region (ISR) (Rossi et al., 2001, Blaiotta et al., 2003).
Most of the studies on the staphylococcal species isolated from dry fermented sausages focused on the flora of the meat products and its evolution along the process. The Staphylococcus house-flora occurring in the environment of processing unit manufacturing naturally fermented sausages has been poorly studied although the environmental flora could contribute to the contamination of the meat products. In this study, we aimed to characterize by molecular methods the staphylococcal flora of a traditional small processing unit through the study of the raw materials, the sausages and the environment. The GCC+ isolates were subjected to a multiplex PCR identifying the ones belonging to the Staphylococcus genus and to the S. xylosus, S. saprophyticus, S. epidermidis and S. aureus species. Staphylococci, which did not belong to the former species, were typed and grouped by the PFGE technique. Then, each PFGE group was identified to species level thanks to the partial sequencing of the sodA gene. Two species S. equorum and S. succinus were found to mainly colonize the meat products and environment of this small processing unit.
Section snippets
Staphylococcal reference species
Strains belonging to the Staphylococcus species described in dry naturally fermented sausages flora were used in this study as reference (Table 1). They were grown at 30 or 37 °C on Brain Heart Infusion (BHI) broth or agar (Difco, Detroit, USA).
Sampling procedures and isolation of strains
A collection of GCC+ strains was obtained by sampling a small unit manufacturing naturally fermented sausages during two seasons, winter and spring. This unit is located in a medium mountain area in France. Dry sausages were manufactured with pork lean
GCC+ counts
The counts and distribution of GCC+ are shown in Fig. 1. All the environmental samples were colonized by the GCC+ flora with counts ranging between 2 × 102 and 1 × 107 CFU/100 cm2. The GCC+ counts did not differ (< 1 log CFU/100 cm2) between the winter and spring samplings but the tables, which were more contaminated in spring. The most colonized environmental samples were the surfaces of drying room and the butcher's block. The counts of GCC+ in the raw materials (fat and lean) were similar in
Discussion
In this study, the diversity of the staphylococcal community of a small unit manufacturing traditional dry fermented sausages was evaluated by the identification and characterization of 412 Staphylococcus strains isolated from the meat products and the environment. Counts of GCC+ colonies dominated by staphylococci (94%) ranged from 1 × 102 to 3 × 105 CFU/g in the batter to 2 × 106 to 2 × 108 CFU/g in the sausages at the end of ripening. The level of GCG+ in the final product was comparable with the
Acknowledgements
This work is part of a FNADT program (1999/3). We are grateful to Jean-Paul Chacornac, Sébastien Masseglia and Brigitte Duclos for technical assistance. The authors would like to thank Jean Labadie for a critical and careful review of the manuscript. S. Corbière Morot-Bizot is the recipient of a fellowship of the French Ministry of “Education Nationale et Recherche”.
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